Quantitative Transcript Profiling Reveals Down-Regulation of A Sterol Pathway Relevant Gene and Overexpression of Artemisinin Biogenetic Genes in Transgenic Artemisia annua Plants

Abstract

To investigate the dynamic fluctuation of terpenoid relevant transcriptomics in transgenic Artemisia annua plants that express the genomic integrated antisense squalene synthase gene (asSS), we have quantified the transcript levels of the sterol anabolic SS gene as well as artemisinin biogenetic amorphadiene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 reductase (CPR) genes by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). The SS mRNA level in transgenic plants sharply droped to 7.4 % – 55.3 % (i. e., 44.7 – 92.6 % reduction as the wild-type control), strongly implying that the expression of endogenous SS gene is significantly suppressed by the exogenous asSS gene. In a synchronous fashion, ADS, CYP71AV1 and CPR mRNA levels elevated with the decline of SS mRNA level in transgenic plants, and the maximal ADS, CYP71AV1 and CPR mRNA levels in transgenic plants were 3.0-, 4.4- and 2.5-fold, respectively, higher than those in the control. Without a lethal effect but with a distinguishable impact on the organogenesis and morphology of transgenic plants, the down-regulation of SS gene has also led to the coordinated overexpression of ADS, CYP71AV1 and CPR genes together with the overproduction of artemisinin although no fully perfect correlation among the available experimental data has been shown.

Abbreviations

ADS:amorphadiene synthase

asSS:anti-sense squalene synthase gene

CPR:cytochrome P450 reductase

CYP71AV1:cytochrome P450 monooxygenase

FPP:farnesyl diphosphate

HPLC:high-performance liquid chromatography

MVA:mevalonate

RFQ-PCR:real-time fluorescent quantitative polymerase chain reaction

SS:squalene synthase

Artemisia annua - Asteraceae - expression profiling - quantification - artemisinin - transgenic plant

References

• 1 Ferreira J FS, Simon J E, Janick J. Relationship of artemisinin content of tissue cultured, greenhouse grown, and field grown plants of Artemisia annua.  Planta Med. 1995;  61 351-5
• 2 Van Geldre E, Vergauwe A, Van den Eeckhout E. State of the art of the production of the antimalarial compound artemisinin in plants.  Plant Mol Biol. 1997;  33 199-209
• 3 Chen D H, Chen J L, Ye H C, Li G F, Liu B Y, Meng Y L. et al . Ri-mediated transformation of Artemisia annua with a recombinant farnesyl diphosphate synthase gene for artemisinin production.  Plant Cell Tiss Org Cult. 1999;  57 157-62
• 4 Chen D H, Ye H C, Li G F. Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation.  Plant Sci. 2000;  155 179-85
• 5 Wallaart T E, van Uden W, Lubberink H G, Woerdenbag H J, Pras N, Quax W J. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin.  J Nat Prod. 1999;  62 430-3
• 6 Wallaart T E, Bouwmeester H J, Hille J. Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.  Planta. 2001;  212 460-5
• 7 Akhila A, Thakur R S, Popli S P. Biosynthesis of artemisinin in Artemisia annua.  Phytochemistry. 1987;  16 1927-30
• 8 Woerdenbag H J, Bos R, Salomons M C. Volatile constituents of Artemisia annua L.  Flavour Fragrance J. 1993;  8 131-7
• 9 Bertea C M, Freije J R, van der Woude H, Verstappen F W, Perk L, Marquez V. et al . Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.  Planta Med. 2005;  71 0-7
• 10 Lommen W JM, Elzinga S, Bouwmeester H J, Verstappen F WA. Artemisinin and sesquiterpene precursors in dead and green leaves of Artemisia annua L. crops.  Planta Med. 2007;  73 1133-9
• 11 Bouwmeester H J, Wallaart T E, Janssen M H, van Loo B, Jansen B J, Posthumus M A. et al . Amorpha-4,11-diene synthase catalyze the first probable step in artemisinin biosynthesis.  Phytochemistry. 1999;  52 843-54
• 12 Ro D K, Paradise E M, Ouellet M, Fisher K J, Newman K L, Ndungu J M. et al . Production of the antimalarial drug precursor artemisinic acid in engineered yeast.  Nature. 2006;  440 940-3
• 13 Mercke P, Bengtsson M, Bouwmeester H J, Posthumus M A, Brodelius P E. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Arch Biochem Biophys 381: 173-80
• 14 Chappell J. The biochemistry and molecular biology of isoprenoid metabolism.  Plant Physiol. 1995;  107 1-6
• 15 Kudakasseril G J, Lam L, Staba J. Effect of sterol inhibitors on the incorporation of ¹⁴C-isopentenyl pyrophosphate into artemisinin by a cell-free system from Artemisia annua tissue cultures and plants.  Planta Med. 1987;  28 280-4
• 16 Vergauwe A, Cammaert R, Vandenberghe D, Genetello C, Inze D, van Montagu M. et al . Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants.  Plant Cell Rep. 1996;  15 929-33
• 17 Feng L L, Yang R Y, Yang X Q, Zeng Q P. Expression of CodA gene from Escherichia coli conferring a negative selective phenotype on transgenic Artemisia annua.  Chin Trad Herb Drugs. 2005;  36 578-82
• 18 Yin L L, Zhao C, Huang Y, Yang R Y, Zeng Q P. Abiotic stress-induced expression of artemisinin biosynthesis genes in Artemisia annua L.  Chin J Appr Environ Bio,. 2008;  14 1-5
• 19 Chappell J. The biochemistry and molecular biology of isoprenoid metabolism.  Plant Physiol. 1995;  107 1-6
• 20 Feng L L, Yang R Y, Yang X Q, Zeng Q P. Gene targeting of squalene synthase in Artemisia annua.  Chin Trad Herb Drugs. 2006;  37 1857-61
• 21 Kanduri C, Thakur N, Pandey R R. The length of the transcript encoded from the Kcnq1ot1 antisense promoter determines the degree of silencing.  EMBO J. 2006;  25 2096-106
• 22 Devarenne T P, Shin D H, Back K, Yin S H, Chappell J. Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor.  Arch Biochem Biophys. 1998;  349 205-15
• 23 Threlfall D R, Whitehead I M. Coordinated inhibition of squalene synthetase and induction of enzymes of sesquiterpenoid phytoalexin biosynthesis in cultures of Nicotiana tabacum. .  Phytochemistry. 1988;  27 2567-80
• 24 Vogeli U, Chappell J. Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor.  Plant Physiol. 1988;  88 1291-6
• 25 Zeng Q P, Zhao C, Yin L L, Yang R Y, Zeng X M, Huang Y. et al . Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification on low temperature-induced gene overexpression.  Sci China Ser C (Life Sci). 2008;  51 232-44